The interleukin-11 receptor variant p.W307R results in craniosynostosis in humans.

Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients.

Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders.

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C>T, BICD2: c.2156A>T, ALS2: c.2171-3T>G, ALS2: c.3145T>A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T>G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.

NGS-driven molecular diagnosis of heterogeneous hereditary neurological disorders reveals novel and known variants in disease-causing genes.

Hereditary neurological disorders (HNDs) are a clinically and genetically heterogeneous group of disorders. These disorders arise from the impaired function of the central or peripheral nervous system due to aberrant electrical impulses. More than 600 various neurological disorders, exhibiting a wide spectrum of overlapping clinical presentations depending on the organ(s) involved, have been documented. Owing to this clinical heterogeneity, diagnosing these disorders has been a challenge for both clinicians and geneticists and a large number of patients are either misdiagnosed or remain entirely undiagnosed. Contribution of genetics to neurological disorders has been recognized since long; however, the complete picture of the underlying molecular bases are under-explored. The aim of this study was to accurately diagnose 11 unrelated Pakistani families with various HNDs deploying NGS as a first step approach. Using exome sequencing and gene panel sequencing, we successfully identified disease-causing genomic variants these families. We report four novel variants, one each in, ECEL1, NALCN, TBR1 and PIGP in four of the pedigrees. In the rest of the seven families, we found five previously reported pathogenic variants in POGZ, FA2H, PLA2G6 and CYP27A1. Of these, three families segregate a homozygous 18 bp in-frame deletion of FA2H, indicating a likely founder mutation segregating in Pakistani population. Genotyping for this mutation can help low-cost population wide screening in the corresponding regions of the country. Our findings not only expand the existing repertoire of mutational spectrum underlying neurological disorders but will also help in genetic testing of individuals with HNDs in other populations.

A Homozygous AKNA Frameshift Variant Is Associated with Microcephaly in a Pakistani Family.

Primary microcephaly (MCPH) is a prenatal condition of small brain size with a varying degree of intellectual disability. It is a heterogeneous genetic disorder with 28 associated genes reported so far. Most of these genes encode centrosomal proteins. Recently, AKNA was recognized as a novel centrosomal protein that regulates neurogenesis via microtubule organization, making AKNA a likely candidate gene for MCPH. Using linkage analysis and whole-exome sequencing, we found a frameshift variant in exon 12 of AKNA (NM_030767.4: c.2737delG) that cosegregates with microcephaly, mild intellectual disability and speech impairment in a consanguineous family from Pakistan. This variant is predicted to result in a protein with a truncated C-terminus (p.(Glu913Argfs*42)), which has been shown to be indispensable to AKNA's localization to the centrosome and a normal brain development. Moreover, the amino acid sequence is altered from the beginning of the second of the two PEST domains, which are rich in proline (P), glutamic acid (E), serine (S), and threonine (T) and common to rapidly degraded proteins. An impaired function of the PEST domains may affect the intracellular half-life of the protein. Our genetic findings compellingly substantiate the predicted candidacy, based on its newly ascribed functional features, of the multifaceted protein AKNA for association with MCPH.

A 24-generation-old founder mutation impairs splicing of RBBP8 in Pakistani families affected with Jawad syndrome.

Jawad syndrome is a multiple congenital anomaly and intellectual disability syndrome with mutation in RBBP8 reported only in two families. Here, we report on two new families from Pakistan and identified a previously reported variant in RBBP8, NM_002894.3:c.1808-1809delTA. We could show that this mutation impairs splicing resulting in two different abnormal transcripts. Finally, we could verify a shared haplotype among all four families and estimate the founder event to have occurred some 24 generations ago.

Updates on Clinical and Genetic Heterogeneity of ASPM in 12 Autosomal Recessive Primary Microcephaly Families in Pakistani Population.

Microcephaly (MCPH) is a genetically heterogeneous disorder characterized by non-progressive intellectual disability, small head circumference, and small brain size compared with the age- and sex-matched population. MCPH manifests as an isolated condition or part of another clinical syndrome; so far, 25 genes have been linked with MCPH. Many of these genes are reported in Pakistani population, but due to a high rate of consanguinity, a significant proportion of MCPH cohort is yet to be explored. MCPH5 is the most frequently reported type, accounting for up to 68.75% alone in a genetically constrained population like Pakistan. In the current study, whole exome sequencing (WES) was performed on probands from 10 families sampled from South Waziristan and two families from rural areas of the Pakistani Punjab. Candidate variants were validated through Sanger sequencing in all available family members. Variant filtering and in silico analysis identified three known mutations in ASPM, a MCPH5-associated gene. The founder mutation p.Trp1326* was segregating in 10 families, which further confirmed the evidence that it is the most prominent mutation in Pashtun ethnicity living in Pakistan and Afghanistan. Furthermore, the previously known mutations p.Arg3244* and p.Arg1019* were inherited in two families with Punjab ethnic profile. Collectively, this study added 12 more families to the mutational paradigm of ASPM and expanded the Pakistani MCPH cohort.

CDK12 inhibition reduces abnormalities in cells from patients with myotonic dystrophy and in a mouse model.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

CDK5RAP2 interaction with components of the Hippo signaling pathway may play a role in primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by a substantial reduction in brain size but with normal architecture. It is often linked to mutations in genes coding for centrosomal proteins; however, their role in brain size regulation is not completely understood. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a novel mutation (XM_011518861.1; c.4114C > T) in CDK5RAP2, the gene associated with primary microcephaly-3 (MCPH3), leading to a premature stop codon (p.Arg1372*). CDK5RAP2 is a component of the pericentriolar material important for the microtubule-organizing function of the centrosome. Patient-derived primary fibroblasts had strongly decreased CDK5RAP2 amounts, showed centrosomal and nuclear abnormalities and exhibited changes in cell size and migration. We further identified an interaction of CDK5RAP2 with the Hippo pathway components MST1 kinase and the transcriptional regulator TAZ. This finding potentially provides a mechanism through which the Hippo pathway with its roles in the regulation of centrosome number is linked to the centrosome. In the patient fibroblasts, we observed higher levels of TAZ and YAP. However, common target genes of the Hippo pathway were downregulated as compared to the control with the exception of BIRC5 (Survivin), which was significantly upregulated. We propose that the centrosomal deficiencies and the altered cellular properties in the patient fibroblasts can also result from the observed changes in the Hippo pathway components which could thus be relevant for MCPH and play a role in brain size regulation and development.

Abolished InsP3R2 function inhibits sweat secretion in both humans and mice.

There are 3 major sweat-producing glands present in skin; eccrine, apocrine, and apoeccrine glands. Due to the high rate of secretion, eccrine sweating is a vital regulator of body temperature in response to thermal stress in humans; therefore, an inability to sweat (anhidrosis) results in heat intolerance that may cause impaired consciousness and death. Here, we have reported 5 members of a consanguineous family with generalized, isolated anhidrosis, but morphologically normal eccrine sweat glands. Whole-genome analysis identified the presence of a homozygous missense mutation in ITPR2, which encodes the type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2), that was present in all affected family members. We determined that the mutation is localized within the pore forming region of InsP3R2 and abrogates Ca2+ release from the endoplasmic reticulum, which suggests that intracellular Ca2+ release by InsP3R2 in clear cells of the sweat glands is important for eccrine sweat production. Itpr2-/- mice exhibited a marked reduction in sweat secretion, and evaluation of sweat glands from Itpr2-/- animals revealed a decrease in Ca2+ response compared with controls. Together, our data indicate that loss of InsP3R2-mediated Ca2+ release causes isolated anhidrosis in humans and suggest that specific InsP3R inhibitors have the potential to reduce sweat production in hyperhidrosis.

Autosomal recessive transmission of a rare KRT74 variant causes hair and nail ectodermal dysplasia: allelism with dominant woolly hair/hypotrichosis.

Pure hair and nail ectodermal dysplasia (PHNED) comprises a heterogeneous group of rare heritable disorders characterized by brittle hair, hypotrichosis, onychodystrophy and micronychia. Autosomal recessive (AR) PHNED has previously been associated with mutations in either KRT85 or HOXC13 on chromosome 12p11.1-q14.3. We investigated a consanguineous Pakistani family with AR PHNED linked to the keratin gene cluster on 12p11.1 but without detectable mutations in KRT85 and HOXC13. Whole exome sequencing of affected individuals revealed homozygosity for a rare c.821T>C variant (p.Phe274Ser) in the KRT74 gene that segregates AR PHNED in the family. The transition alters the highly conserved Phe274 residue in the coil 1B domain required for long-range dimerization of keratins, suggesting that the mutation compromises the stability of intermediate filaments. Immunohistochemical (IHC) analyses confirmed a strong keratin-74 expression in the nail matrix, the nail bed and the hyponychium of mouse distal digits, as well as in normal human hair follicles. Furthermore, hair follicles and epidermis of an affected family member stained negative for Keratin-74 suggesting a loss of function mechanism mediated by the Phe274Ser substitution. Our observations show for the first time that homozygosity for a KRT74 missense variant may be associated with AR PHNED. Heterozygous KRT74 mutations have previously been associated with autosomal dominant woolly hair/hypotrichosis simplex (ADWH). Thus, our findings expand the phenotypic spectrum associated with KRT74 mutations and imply that a subtype of AR PHNED is allelic with ADWH.

Evidence for autosomal recessive inheritance in SPG3A caused by homozygosity for a novel ATL1 missense mutation.

Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of disorders characterized by progressive spasticity and weakness of the lower limbs. Autosomal dominant and 'pure' forms of HSP account for ∼80% of cases in Western societies of whom 10% carry atlastin-1 (ATL1) gene mutations. We report on a large consanguineous family segregating six members with early onset HSP. The pedigree was compatible with both autosomal dominant and autosomal recessive inheritance. Whole-exome sequencing and segregation analysis revealed a homozygous novel missense variant c.353G>A, p.(Arg118Gln) in ATL1 in all six affected family members. Seven heterozygous carriers, five females and two males, showed no clinical signs of HSP with the exception of sub-clinically reduced vibration sensation in one adult female. Our combined findings show that homozygosity for the ATL1 missense variant remains the only plausible cause of HSP, whereas heterozygous carriers are asymptomatic. This apparent autosomal recessive inheritance adds to the clinical complexity of spastic paraplegia 3A and calls for caution using directed genetic screening in HSP.

Novel missense mutation in the RSPO4 gene in congenital hyponychia and evidence for a polymorphic initiation codon (p.M1I).

BACKGROUND: Anonychia/hyponychia congenita is a rare autosomal recessive developmental disorder characterized by the absence (anonychia) or hypoplasia (hyponuchia) of finger- and/or toenails frequently caused by mutations in the R-spondin 4 (RSPO4) gene. METHODS: Three hypo/anonychia consanguineous Pakistani families were ascertained and genotyped using microsatellite markers spanning the RSPO4 locus on chromosome 20p13. Mutation screening of the RSPO4 gene was carried out by direct sequencing of the entire coding region and all intron-exon boundaries. RESULTS: Mutations in the RSPO4 gene were identified in all families including a novel missense mutation c.178C>T (p.R60W) and two recurrent variants c.353G>A (p.C118Y) and c.3G>A (p.M1I). The c.3G>A variant was identified in unaffected family members and a control sample in a homozygous state. CONCLUSIONS: This study raises to 17 the number of known RSPO4 mutations and further expands the molecular repertoire causing hypo/anonychia. The c.353G>A emerges as a recurrent change with a possible founder effect in the Pakistani population. Our findings suggest that c.3G>A is not sufficient to cause the disorder and could be considered a polymorphism.

Non-bullous congentital ichthyosiform erythroderma associated with homozygosity for a novel missense mutation in an ATP binding domain of ABCA12.

A Mutations in the gene encoding the ABCA12 protein are associated with different subtypes of autosomal recessive congenital ichthyosis (ARCI), including Harlequin ichthyosis (HI), lamellar ichthyosis (LI) and non-bullous congenital ichthyosiform erythroderma (NCIE). Disruption of ABCA12 lead to perturbed lipid transport in lamellar granules and a defective intercellular lipid layer of the stratum corneum. We have identified a large consanguineous Pakistani family affected by NCIE. Autozygosity mapping showed that affected individuals are homozygous for the ABCA12 gene region. Subsequent mutation screening revealed a homozygous c.4676G>T transition in all five affected family members. The mutation results in a novel p.G1559V substitution within the first nucleotide binding domain of ABCA12. The combined results support that an ABCA12 missense mutation, despite its location in a functional domain, may be associated with a mild ichthyosis phenotype. Furthermore, our findings increase the mutational spectrum in ABCA12 associated with ARCI of diagnostic and prognostic importance.

Autozygosity mapping of a large consanguineous Pakistani family reveals a novel non-syndromic autosomal recessive mental retardation locus on 11p15-tel.

Autosomal recessive inherited mental retardation is an extremely heterogeneous disease and accounts for approximately 25% of all non-syndromic mental retardation cases. Autozygosity mapping of a large consanguineous Pakistani family revealed a novel locus for non-syndromic autosomal recessive mental retardation (NS-ARMR). The affected individuals showed low IQ and cognitive impairment without any neurological, skeletal, and biochemical abnormalities. All known NS-ARMR genes were excluded by STS markers, so autozygosity mapping by microarray single-nucleotide polymorphism (SNP) analysis were done in all sampled individuals in the family. The nuclear central loop in the five generation family showed homozygosity for a 6-Mb telomeric region on 11p15, whereas all other linkage regions were excluded by calculation of logarithm of odds (LOD) for the SNP microarray data. A maximum LOD score of Z = 3.31 was calculated for the mapped region. These results suggest a novel genetic locus, MRT17, for NS-ARMR.